Cell washing device and method

ABSTRACT

A cell washer is disclosed. The cell washer includes a vessel configured to hold cells. The vessel includes an elongated body including an opening, an inner surface, and a pocket defined by a first inner surface portion of the inner surface disposed between and radially outward relative to a second inner surface portion and a third inner surface portion of the inner surface, and a cavity. The vessel also includes an actuating device capable of causing the vessel to spin about an axis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application of U.S. Application No.16/468,487, which is a PCT National Stage of International PatentApplication No. PCT/US2017/066312, filed on Dec. 14, 2017, which claimspriority to U.S. Provisional Application No. 62/434,718, filed on Dec.15, 2016, the disclosures of which are hereby incorporated by referencein their entireties. To the extent appropriate a claim of priority ismade to each of the above-disclosed applications.

TECHNICAL FIELD

Embodiments of the invention relate to the field of particlepreparation, and more particularly to devices and methods for washingcells.

BACKGROUND

Particle analysis generally, and cellular analysis in particular,frequently requires removing or replacing liquids that suspend particlesor cells. This is useful to remove interfering substances, excessstains, unbound labeled antibodies, detergents, permeabilizing agents,lysing agents, fixatives, neutralizing agents, and other materials. Insome situations, cell washing may also be useful to reduce unwantedinteractions between cell types mediated by dissolved materials.

Traditional methods for particle or cell washing involve a variety ofprocesses such as sedimenting and decanting, acoustic separation,centrifugation, filtration, flow through structured channels, andmagnetic separation. These methods are difficult to automate, bulky,time-consuming, and frequently need single-use consumables. There is athus a need for an easily automated process and device for cell washing.

A purpose of cell washing is to remove unwanted substances that mayaffect further processing or subsequent analysis. Cell washing generallyinvolves removal of a suspending liquid and resuspension of the cells ina replacement liquid, generically called a wash liquid. Cell washing mayuse different wash liquids at different portions of the wash process andmay also include extended exposure to selected wash liquids or agitationto encourage transfer of unwanted substances.

Cell washing may lose, injure, activate, alter, or destroy cells, maycause undesirable interaction between cells, or may differentiallydeplete a sample of certain cell types. These effects can alter theresults of subsequent analysis. For example, exposure of cells to highaccelerations (such as that produced by inertial effects of high-speedrotation or “centrifugal force”) or high fluid shear rate can rupturesome cell types more than others. An assay of a washed cell mixture thusmay not accurately reflect the proportions of cells present in theoriginal sample. There is a thus a need for a process and device forcell washing that preserves the properties and proportions of a widevariety of cells in a sample.

Wash efficacy is one measure of the quality of a wash procedure. A washhas a high efficacy if it removes more of the unwanted substances. Aprocess with low wash efficacy leaves more of the unwanted substances.Multiple instances of a process may in some cases be chained to improvethe wash efficacy of the combined process. A simple dilution model,though not realistic for all wash processes, serves to illustrate theconcept of wash efficacy. If a wash process leaves at the end of thewash a fraction d of the fluid present at the start of a wash, thenafter n iterations, the fraction of the original fluid (including theoriginal unwanted substances) remaining is (d)^(n). The wash efficacymay be expressed as the inverse of the fraction remaining: in this case⅟d for a single process and ⅟d^(n) for the multiple iteration process.For example, if a wash process leaves 10% of the original liquid afterone iteration, its wash efficacy may be expressed as ⅟0.1 = 10. Fouriterations of such a wash process gives an ideal combined wash efficacyof ⅟0.1⁴ = 10,000.

However, wash steps may not be chained without consequence: additionalwash iterations take additional time and may increase the likelihood ofcellular alteration or loss. Further, the resuspension process may beparticularly damaging to cells as it typically relies upon high fluidshear. There is a thus a need for a process and device for cell washingthat efficaciously washes cells with fewer wash iterations and hencefewer instances of resuspension.

Cells may be sensitive to the magnitude and duration of applied forcesduring washing; extended exposure to high forces causes more damage tocells. However, in centrifugal cell washers, the time for cells tosediment under an applied force decreases as the force increases. Thereis thus a need for a cell washer device and process that reduces theexposure of cells to high forces for extended times.

Living cells are generally slightly denser than aqueous wash liquids. Incentrifugal cell washers, the denser cells sediment through the washliquids in regions of high relative force. A wash protocol in acentrifugal cell washer usually includes repeated cycles of sedimentingthe cells, removing as much as possible of the wash liquid, andresuspending the cells in fresh wash liquid. The time required tosediment cells depends on the distance the cells travel through the washliquid and is hence longer with larger volumes of wash liquids presentbefore sedimentation. It is therefore desirable to sediment from aninitially small volume of wash liquid. The wash efficacy of such aprocess is limited by the amount of wash liquid remaining after eachremoval step (an amount usually limited by mechanical constraints) ascompared to the total volume of wash liquid exposed to the cells. Thusit would be beneficial to sediment the cells from an initial low volumeof wash liquid but to expose the cells to additional wash liquid aftersedimentation. However, once the cells are sedimented to a region ofhigh relative force, additional wash liquid cannot appreciably interactwith cells because the force causes the less dense liquid to float“above” the cells. There is thus a need for a cell washer device andprocess that permit interaction of additional wash liquid with cellsundergoing wash during a single wash iteration.

In centrifugal cell washing, use of closed containers requires complexrotating connections to add or remove liquids. However open containerspermit liquids to escape unless the container volume is large comparedto the contained volume. There is thus a need for an open-container cellwasher that accommodates relatively large volumes of liquids.

Embodiments of the invention solve these and other problems individuallyand collectively.

SUMMARY

In some embodiments, the invention includes a cell washer having avessel to hold cells. The vessel includes an elongated body defining anopening, a cavity, and a pocket. The opening communicates with thecavity, and the cavity communicates with the pocket. The pocket canextend radially outward relative to the portion of the elongated bodydefining the cavity. The pocket can be symmetric about a verticallyoriented axis. The elongated pocket has a radial depth that extendsbeyond the side walls forming the cavity. The pocket can have an aspectratio (e.g., length to depth, when viewed from an axial cross-section)of about 2:1, 4:1, 10:1, or 15:1 or more. An actuating device such as arotor can spin the vessel about the axis.

The cavity may be defined at least in part by a cylindrical section ofthe body and may be positioned below the pocket. The inner surfacedefining forming the cavity may transition to the pocket as asigmoid-shaped transition.

The cell washer may also include a conduit that passes through theopening and into the cavity. The conduit is configured to transfer fluidto and from the cavity during rotation of the vessel. The conduit endsin a tip that may be disposed adjacent an interior wall of the vesseland below the pocket. The conduit may be disposed off of the axis andparallel to the axis. The conduit may be fluidically coupled to a fluidpump.

The vessel may also include an upper annular region defined by an upperportion of the body. The upper annular region may be disposed betweenthe pocket and the opening.

In some embodiments, the cell washer may also include a probe mounted onan elevator with the elevator configured to lower the probe through theopening. A sample pump may be in fluid communication with the probe. Thepump and probe allow for the addition or removal of material from thevessel. A controller can be electrically connected to the actuatingdevice (e.g., rotor), the fluid pump, the sample pump, and the elevatorcan control the operation of these components.

Embodiments of the invention the invention also include a methodincluding washing cells from a sample including cells suspended in aliquid. The method has steps of dispensing the sample into a vesselincluding a cavity and a pocket, where the cavity and the pocket aredisposed about an axis that is vertically oriented. The pocket can bepositioned radially outward with respect to a portion of the bodyforming the cavity. Other steps include rotating the vessel at a firstspeed about the vessel’s axis, displacing the cells into the pocket,sedimenting the cells toward the wall of the pocket, and withdrawing atleast part of the liquid.

The step of displacing the cells into the pocket includes adding a firstaliquot of a wash liquid to the vessel. The wash liquid is added througha conduit extending into the vessel and terminating below the pocket.The step of withdrawing includes aspirating through the same conduit ata different time.

Other steps may include adding a second or subsequent aliquot of a washliquid through the conduit and resuspending the cells. In someembodiments, the steps of withdrawing at least part of the liquid fromthe vessel and of adding a second (or subsequent) aliquot of wash liquidmay be repeated one or more times to provide a more efficacious wash.For example, the steps can be repeated between 2 and 5 times in someembodiments of the invention.

The step of rotating the vessel at the first speed produces a force inthe pocket of at least about 100, or 250 x g. The method of washingcells may also include rotating the vessel at a second speed during thewithdrawing step. This rotation at a second speed may produce a force inthe pocket of at least about 25 x g. Thus, in some embodiments, thesubsequent vessel rotation speeds can decrease with each successive washstep.

The step of resuspending the cells may include sub-steps of adding aresuspension liquid, and stopping the rotation. In some embodiments, therotation may be abruptly reversed in direction to assist in resuspendingthe cells. Washed cells may be removed once the rotation is stopped.

The volume of the sample may be less than the volume of the cavity belowthe pocket, and in some embodiments, may be less than about 0.7 timesthis volume below the pocket. The volume of the resuspension liquid maybe less than the volume of the sample so that, after washing, the washedcells are suspended at a higher concentration than were the cells in thesample before the wash.

The method may include additional steps to clean the vessel forsubsequent use, including steps of adding a rinse liquid, rotating thevessel at a third speed, and aspirating the rinse liquid through theconduit. The step of cleaning the vessel may include varying the speedof rotation during the step of aspirating the rinse liquid.

The wash liquid or the resuspension liquid may include an suitablematerial. For example, it may include about 5mM EDTA and may alsoinclude about 0.1 % to about 2% fetal calf serum. In some embodiments,the wash solution is comprised of an isotonic buffer (e.g., PBS), ananticoagulant (e.g., EDTA), and a protein (e.g., fetal bovine serum). Insome embodiments, fetal calf serum can server as the protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B shows diagrammatic side sectional views of an embodimentof the device of the invention.

FIG. 2 shows a diagrammatic side sectional view of the vessel of theembodiment of FIG. 1 .

FIGS. 3A - 3F show diagrammatic steps of an exemplary cell washingprotocol.

FIG. 4 shows a flow chart of the cell washing protocol of FIGS. 3A - 3F.

FIGS. 5A - 5 f show diagrammatic steps of an exemplary vessel cleaningprotocol.

FIG. 6 shows a flow chart of the vessel cleaning protocol of FIGS. 5A -5 f .

FIG. 7 shows scatter plots of white blood cell recovery usingembodiments of the device and method of the invention compared totraditional wash and to no wash.

FIG. 8 shows scatter plots for a five-part differential WBC analysisusing embodiments of the device and method of the invention compared totraditional wash and to no wash.

FIG. 9 shows scatter plots of platelet monocyte interactions usingembodiments of the device and method of the invention compared totraditional wash and to no wash.

FIG. 10 shows cell viability using embodiments of the device and methodof the invention compared to traditional wash and to no wash.

FIG. 11 shows Kappa / Lambda separation using embodiments of the deviceand method of the invention compared to traditional wash.

FIG. 12 shows total cell recovery using embodiments of the device andmethod of the invention compared to traditional wash.

FIG. 13 shows lymphocyte T-cell recovery using embodiments of the deviceand method of the invention compared to traditional wash and to no wash.

FIG. 14 shows B cell and NK cell recovery using embodiments of thedevice and method of the invention compared to traditional wash and tono wash.

FIG. 15 shows a partial side cross-sectional view of an assemblyaccording to an embodiment of the invention.

FIG. 16 shows a top cross-sectional view of the assembly shown in FIG.15 .

DETAILED DESCRIPTION

FIGS. 1A and 1B show diagrammatically an embodiment of the cell washeraccording to an embodiment of the invention. Cell washer 1 includesvessel 10 comprising a body, a rotor 12, conduit 18, and controller 20.In some embodiments, cell washer 1 may also include pipettor 16 andhousing 14.

FIG. 2 shows vessel 10 in more detail. Vessel 10 comprises an elongatedbody that can be configured to contain the cell sample and wash liquidsduring the wash process. Vessel 10 can be oriented with its long axis 42vertical during operation. Vessel 10 includes a closed bottom 36, anopen top 38, and a wall 28 extending between bottom 36 and top 38. Wall28 defines a cavity 30 and a pocket 34.

In this document, the inner surface of the wall 28 can refer to aportion of vessel 10 bounding cavity 30 or pocket 34. The outer surfaceof the wall 28 can refer to a portion of vessel 10 separated from cavity30 or pocket 34 by some portion of wall 28.

In this document, pocket 34 is denoted separately from cavity 30 eventhough these two features are defined by an inner surface of a body ofthe vessel 10. The inner surface of the body may include a first innersurface portion 37A that disposed between and radially outward relativeto a second inner surface portion 37B and a third inner surface portion37C to form pocket 34. Cavity 30, which may taper towards axis 42, isdifferentiated from pocket 34 by the respective radii of their definingsurface portions 37A, 37B. For example, a first inner surface portion37A may define at least part of the pocket 34, while a second innersurface portion 37B may define at least part of the cavity. As show inFIG. 2 , the first inner surface portion 37A is further away (in aradial sense) from the long axis 42 than the second inner surfaceportion 37B. In the embodiment in FIG. 2 , the pocket radius of thebottom of the pocket 34 (which corresponds to a middle inner sidewall ofthe body of the vessel 10) is substantially constant (other thantransition zones at one or both ends of pocket 34, the transition zonesbeing between the first and second inner surface portions 34A, 34B, andthe first and third inner portions 34A, 34C) over the axial extent ofpocket 34. The radius of the bottom of the pocket 34 may also beconstant over most or all of its axial extent, in which case the spaceformed by the pocket 34 may form a cylindrical shell of definedthickness (the depth of the pocket). The hollow or void inside of vessel10 may include cavity 30 as well as an upper annular region 39, whichmay be defined by an upper section 40 of the body of the vessel 10. Theextent of pocket 34 includes any transition zone that may be small (lessthan about one tenth the axial extent of the pocket) with respect to theaxial extent of the pocket. The aspect ratio of the pocket may begreater than about 2:1, 3:1, 5:1, 10:1 or 15:1 (e.g., where the aspectratio is the length: depth). The radius can change between the radius ofthe surrounding areas of cavity 30 and the increased radius of pocket34.

Wall 28 and the body in which the wall 28 is included may be formed ofstable solid materials such as glass, polycarbonate, or acrylic plastic.In some embodiments, use of high density polyethylene improves washefficacy due to reduced adhesion. Transparency is beneficial duringprocess development as fluid behavior and cleanliness may be observedthrough wall 28 during operation.

In some embodiments, vessel 10 and cavity 30 are symmetrical about axis42. The outer surface of wall 28 may be similar in shape to the innersurface of wall 28 so that wall 28 is of relatively constant thickness.This has the benefit of reducing the mass of vessel 10 and thus reducingthe torque required to spin vessel 10. The outer surface of wall 28 (andhence of vessel 10) may include a cylindrical profile and may terminatein a hemispherical bottom end, so that the outer surface of vessel 10 issimilar in shape to a conventional test tube.

In some embodiments, cell washer 1 may be designed to process sampleshaving volume on the order of 1 mL. In such embodiments, cavity 30 mayfit within a cylindrical envelope of about 0.35 inches diameter andabout 2 inches tall. However, in other embodiments, cell washer 1 may beeither larger or smaller for other sample sizes by scaling to maintainforces and relative volumes.

The inner surface (which may correspond to a second inner surfaceportion 37B) of wall 28 can define at least part of a lower portion 32of the vessel as well as the cavity 30. In some embodiments, an upperannular region 39 may be present in an upper portion 40 of the vessel10. Lower portion 32, middle portion 55, and upper portion 40 aredisposed along different axial lengths of vessel 10. Lower portion 32including the cavity 30 extends upward from the inner surface of bottom36. Middle portion includes a central annular region 56 including thepocket 34 and begins above lower portion 32 and extends further upward.Upper portion 40 includes the annular region 39, and when present,begins above central annular region 56 and extends further upward to theinner surface of open top 38. Wall 28 bounding cavity 30 may be formedwith a smooth inner surface, without sharp inside corners to avoidtrapping any cells or liquid during use. The minimum inside cornerradius can depend on the surface properties of the wall material and theability of liquids within vessel 10 to wet that wall material. In someembodiments, using aqueous fluids in polycarbonate vessels, the minimuminside corner radius may be about 0.03 inches. A benefit of separatingpocket 34 from lower portion 32 is that pocket 34 provides a locationwhere centrifugal force confines the particles at a position separatefrom where liquids are added or removed. This prevents unintendedremoval of particles during the wash process, increasing recovery.

Bottom 36 is the closed end of the elongated body that forms vessel 10.Bottom 36 is positioned downward during operation of cell washer 1.Directions in this description refer to this orientation. The outersurface of wall 28 at bottom 36 is the physical bottom of vessel 10. Theinner surface of wall 28 at bottom 36 is the physical bottom of cavity30.

Lower portion 32 begins at the intersection of axis 42 and the innersurface of wall 28 at bottom 36. A purpose of lower portion 32 is toallow selected addition or removal of liquids without affecting thecells. Cells may be retained in pocket 34 under the influence ofcentrifugal force while fluids are added to and removed from lowerportion 32. The inner surface of wall 28 tapers smoothly andmonotonically outward from axis 42 and upward from bottom 36 until itreaches a lower radius 33. Lower radius 33 is the maximal inside radiusof lower portion 32. Lower portion 32 may continue further at lowerradius 33 to form a cylindrical section 31 of lower portion 32 or mayterminate at the transition to pocket 34. In some embodiments, lowerportion 32 is similar in shape to a test tube, with a curved lowerportion and a cylindrical upper portion. In such embodiments, the radiusof the cylindrical upper portion corresponds to lower radius 33. Thecurved lower portion may be substantially hemispherical, parabolic, orany other profile that transitions from the intersection of centerlinewith bottom 36 to pocket radius 35. The volume of lower portion 32 maybe large enough to contain the entirety of the sample containing thecells to be washed. A benefit of lower portion 32 tapering is that theoutwardly directed centrifugal force urges the relatively dense cells orparticles upward along the taper towards pocket 34. A second benefit isthat the liquid flows to the bottom center when rotation stops,improving recovery by probe 54.

Pocket 34 can form part of the central annular region 56 of the regionof the interior of vessel 10 that has the largest internal radius. Apurpose of pocket 34 is to contain the cells during the wash process ina limited volume so that suspending liquids may be interchanged withoutloss of cells. Pocket 34 may have essentially constant inside radius(pocket radius 35) except for where it joins to lower portion 32 (and toupper portion 40 if present). In other embodiments, pocket 34 may haveother shapes. For example, pocket 34 may be slightly ellipsoidal so thatcells preferentially sediment to the “deep” portion of the ellipsoid.This may be useful when the sample has very low cell concentrations.Still other shapes may be useful in other applications.

In embodiments, the inner surface of wall 28 at the boundary of lowerportion 32 and pocket 34 may form a sigmoid-shaped transition 41 (asviewed in an axial cross section as shown in FIG. 2 ). The benefit ofthe sigmoid-shaped transition 41 is that it reduces the trapping orhang-up of particles as they move between pocket 34 and lower portion32. This increases recovery and reduces carryover between samples.

In an embodiment with a cylindrical segment in pocket 34, the activevolume of pocket 34 is the difference between the volume of cavity 30between the ends of pocket 34 and the volume of a cylinder having radiusequal to lower radius 33 between the ends of pocket 34. The activevolume is thus a cylindrical shell with one or two sigmoid-shapedtapering ends. A benefit of the cylindrical segment is that cells may bepacked in a thin layer, making cleaning easier and subjecting cells tosimilar g-forces for a given rotation rate. Another benefit of thecylindrical segment is elimination of trapped volumes of fluid,improving wash efficacy.

In some embodiments, pocket 34 includes an active volume at least equalto the aggregate volume of cells or particles in the sample. In otherembodiments, pocket 34 includes an active volume at least equal to thetotal volume of the sample. This permits the entirety of a sample to fitwithin pocket 34 during cell sedimentation, supporting suchsedimentation at near constant radius (and thus near constant force)irrespective of the fraction of the sample that is cellular. In stillother embodiments, pocket 34 includes an active volume at least equal toabout 1.4 times the total volume of the sample. This greater volumesupports sedimentation at near constant radius even if the sample werediluted during the upward displacement step described in more detailbelow.

Upper portion 40, when present, begins above pocket 34 and extends totop end 38. A purpose of upper portion 40 is to provide a location toaccommodate wash liquid that had traversed pocket 34 so that cells inpocket 34 may be exposed to fresh wash liquid. Upper portion 40 taperssmoothly and monotonically inwardly from pocket 34 and merges with topend 38 so that radial forces tend to force any relatively high densitymaterials into pocket 34. The volume of upper portion 40 may be similarto that of lower portion 32 to receive fluids first supplied to lowerportion 32. In some embodiments, upper portion 40 may have a largervolume than lower portion 32 because the roughly parabolic profile ofthe fluid-free margin consumes a larger fraction of the volume of upperportion 40 than it does of lower portion 34. Upper portion 40 and thelower portion 32 may include cylindrical segments having about the sameradius. A benefit of the paired upper and lower portions surrounding apocket is that they allow wash liquid added in lower portion 32 to sweepthrough the cells confined in pocket 34, rinsing the cells and sweepingthe dirty wash liquid away from the cells. This increases wash efficacywhile minimizing wash liquid volume.

When vessel 10 spins about axis 42, the fluid contents of vessel 10distribute radially outward and upward under the combined influence ofcentrifugal force and gravity. The fluid contents forms a cup-shapedhollow shell bounded by wall 28 on the outside, by bottom 36 below, byannular lip 46 above, and by a free fluid margin centrally. Thefluid-free margin assumes a roughly parabolic shape with parametersdetermined by the speed of rotation, by the surface tension of thefluid, and by the contact angle of the wall material with respect to thefluid. V. A. Lubarda published a detailed analysis of the shape of thefluid-free margin in Acta Mech (2013) 224: 1365, the entirety of whichis hereby incorporated by reference. As rotation speed increases, theparabolic edge of the fluid-free boundary descends toward bottom 36, andmay contact bottom 36 so that the fluid is distributed in an open shell.

Top end 38 has an opening 44 centered on the axis 42 and lip 46surrounding opening 44. A purpose of top end 38 is to contain liquids invessel 10 during rotation. Opening 44 may be circular and define anopening diameter. Opening radius may be selected so that fluid remainswithin vessel 10 at the maximum operating rotation speed. In someembodiments, the maximum operating speed generates a centrifugal forceof about 400 x g and the opening radius is about 0.25 inches. Thediameter of the opening 44 can be large enough to allow the introductionof sample and the removal of fluid, and small enough to keep the fluidfrom escaping at maximum spin speed. In some embodiments, the diameterof opening 44 may be between about 0.157 inches and about 0.25 inches.

A benefit of lip 46 surrounding opening 44 is that lip 46 retains theliquid contents while vessel 10 spins and permits access to non-spinningdevices such as the conduit 18 or pipettor 16. In other embodiments,surface tension and gravity may retain the contents of vessel 10 whilespinning and lip 46 may be absent.

Conduit 18 includes one or more tubes 64, a tip 66, and associatedfluidics. A purpose of conduit 18 is to deliver and remove liquids tovessel 10. Tube 64 has a free end at tip 66 and a coupled end connectedoutside of vessel 10 to associated fluidic components such as abidirectional pump 68, one or more valves 74, one or more wash liquidreservoirs 70, and a waste reservoir 72. The external fluidic componentsare configured to deliver one or more wash liquids and to remove wasteliquids through tube 64 under programmable control. This bidirectionalliquid transfer may be driven by bidirectional pump 68, which may be asingle pump (such as a syringe pump or peristaltic pump) or may be twoor more pumps that each may operate in a single direction. In someembodiments, conduit 18 may be plumbed through housing 14.

Tube 64 enters vessel 10 through opening 44 and extends downwards intolower portion 32. Tube 64 may be a circular tube such as a section ofhypodermic needle stock. Tube 64 may be straight over most of its lengthbut may include a bend near its free end so that tip 66 may bepositioned near the tapered portion of wall 28. The straight portion oftube 64 may be disposed generally parallel to axis 42 but displacedoff-axis (e.g., not co-linear, but parallel) toward one side of annularlip 46. In some embodiments, conduit 18 (including tube 64) does notrotate with vessel 10, so tube 64 is positioned so as not to contact anyportion of vessel 10. The off-axis position of tube 64 allows entry of(the generally larger diameter) pipettor 16 without contacting eithertube 64 or vessel 10. A benefit of tube 64 extending from above vessel10 is that fluids can be added or removed from outside vessel 10. Abenefit of tube 64 extending through opening 44 is that liquid may beadded or removed without rotating seals, reducing system complexity. Abenefit of tube 64 terminating adjacent the inner wall is that thismaximizes the fraction of liquid removable during wash, increasing washefficacy. It also removes the liquid at a lower g-force than thatapplied to particles in the pocket, simplifying the associated fluidics.Benefits of tube 64 terminating below pocket 34 include the benefit thatadded liquid scrubs through the cells in pocket 34 and that no containedcells are removed as liquid is withdrawn. This increases wash efficacyand improves recovery.

The shape and position of tube 64 as it extends through the upperportions of vessel 10 has unexpected effects. For example, tube 64stabilizes liquid within vessel 10 during rotation and affects the shapeof pulses of wash liquid. With tube 64 configured as described above,vessel 10 can accommodate fluid volumes at rotation rates that otherwisewould be expected to overflow vessel 10 through opening 44. Tube 64sculpts the shape of pulses of added wash liquid as they propagatethrough vessel 10 producing a more efficacious wash. Without intent tobe bound by theory, it is believed that the first effect is due tosurface interactions between tube 64 and the parabolic edge of thefluid-free boundary during rotation, and the second effect is due to acombination of surface tension and viscous drag effects. Cell washer 1takes advantage of these effects to manipulate the fill level and therotation rate of vessel 10 so as to send pulses of wash fluid upwardsand downwards through cells sedimented in pocket 34.

The bend near tip 66 permits tube 64 to remain in its vertical off-axisposition over most of its length, while permitting positioning of tip 66in a desired location further away from axis 42. The bend may positiontip 66 to within less than about 0.06 inches from wall 28 to aspirateand deliver liquids during processing. The bend may form any suitableangle including 90, 120, 130, etc. degrees. This relatively largedistance reduces tolerances in the manufacture of cell washer 1. Inembodiments, tip 66 may be within less than about 0.03 inches from wall28 to allow conduit 18 to aspirate a larger fraction of liquid remainingafter a wash phase and thereby increase wash efficacy. The section ofwall 28 proximate tip 66 may be within the tapered portion of lowerportion 32. This position permits addition of liquid during rotationthat displaces sample components upwards into pocket 34 and removal ofliquids with disturbing cells sedimented in pocket 34.

Rotor 12 spins vessel 10 about axis 42. Rotor 12 includes motor 48, andcoupling 52. The purpose of rotor 12 is to develop controlledcentrifugal force in vessel 10. Motor 48 may be any of a variety ofmotors known in the art, such as a brushless DC motor. In someembodiments, motor 48 is capable of spinning vessel 10 at a speed of atleast about 10000 rotations per minute to develop a radial force ofabout 400 x g or more. Appropriate bearings support the spinning parts.

Motor 48 is electrically connected to controller 20. Controller 20provides electrical signals as required to motor 48 through a driver(not shown). Coupling 52 connects motor 48 to vessel 10. Coupling 52 maybe integrally formed with vessel 10, such as a hole in the outside ofbottom 36 formed to accommodate a keyed or pinned shaft of motor 48.Alternatively, coupling 52 may include a hollow that accommodates theoutside of vessel 10. In such embodiments, coupling 52 may includewindows to afford a view of the process during operation.

Although a rotor 12 is shown for purposes of illustration, any othersuitable actuating device may be used. For example, instead of a rotor,the vessel 10 could have magnets within it, which may beelectromagnetically coupled to coils in a surrounding container suchthat the coils and a corresponding electrical source may cause thevessel 10 to move by electromotive force.

Pipettor 16 includes probe 54, elevator 56, and sample pump 58. Apurpose of pipettor 16 is to add samples to be washed and to removewashed cells from vessel 10. Pipettor 16 may also remove rinse fluidafter vessel cleaning.

Probe 54 can be an elongated tube that enters through opening 44 whilerotation is stopped. The purpose of probe 54 is to contain and deliverliquids to and from vessel 10. Probe 54 may be a washable tube such asthose commonly employed in chemistry or hematology analyzers.Alternatively, probe 54 may be a disposable pipette tip coupled to apipettor mandrel. Probe 54 may also include conventional level sensingdevices, such as a capacitive level sensor, to detect the level offluids within sample containers and in vessel 10.

Probe 54 approaches vessel 10 at or near the bottom center to assuremaximal removal of material. Probe 54 enters vessel 10 closer to thevessel axis than tube 64 is disposed to avoid collisions. The benefitsof using a probe separate from conduit 18 to add and remove liquid isthat probe 54 can pipette sample, reducing carryover compared to themore complex fluidic connections of conduit 18, and that the probe canapproach the bottom of the vessel without risk of disturbing theposition of tip 66.

Elevator 56 may be constructed of conventional positioning componentsincluding motors and slides. Its purpose is to raise and lower probe 54through opening 44 to deliver or remove liquids. Elevator 56 may alsoinclude at least one additional axis of motion to position probe 54 withrespect to sample containers, waste receptacles, probe washers, orliquid reservoirs. Sample pump 58 is fluidically coupled to probe 54 andsupplies motive force to move fluids within probe 54. Sample pump 58 maybe any of a variety of conventional pumps capable of deliveringcontrolled volumes of sample, such as a syringe pump or a piston pump.

In other embodiments, conduit 18 may perform some or all of thefunctions of pipettor 16. In such embodiments, conduit 18 may furtherinclude a conduit transport (not shown) that positions tube 64 withrespect to vessel 10. The conduit transport may be constructed ofconventional positioning components including motors and slides.

Some embodiments of cell washer 1 may also include a housing 14. Housing14 surrounds vessel 10 and may also surround portions of rotor 12, andconduit 18 as illustrated in FIG. 1A. A purpose of housing 14 is tocontain any leaked or aerosolized materials resulting from the washprocess. Housing 14 may couple fluidically to conduit 18 so that conduit18 is plumbed to bidirectional pump 68 through a portion of housing 14.Housing 14 may also include a hole aligned with the axis of vessel 10 toallow entry of probe 54. In some embodiments housing 14 also includes avacuum port attachable to a suction pump to remove aerosolized material.

Controller 20 may be a conventional controller such as a microcomputer,microprocessor, programmable logic controller, or similar device capableof flexibly sequencing operations of the rotor, elevator, and fluidicsto perform one of a variety of cell washing protocols. In operation, auser may select a stored protocol or may compile steps for a newprotocol that controller 20 will subsequently execute. Controller 20typically controls mechanical devices such as elevator 56, rotor 12,bidirectional pump 68, sample pump 58, and other components bygenerating low level signals. Drivers (not shown) may translate the lowlevel signals to drive signals appropriate to each mechanical device. Insome embodiments, controller 20 may also receive signals from devicesreporting the status of cell washer 1. Such devices may includetachometers or encoders for rotation feedback, level sensors forpipettor or conduit feedback, and video signals for separation progressfeedback, among others.

In some embodiments, the controller 20 may be embodied by a processorand a computer readable medium coupled to the processor. The computerreadable medium may comprise code, executable by the processor, forimplementing any of the functions described herein.

Cell Wash and Vessel Cleaning

The sample may include whole blood treated with red cell lysing agent,or any other cellular sample where a wash is desired. Wash liquids maybe isotonic buffers, which may in some embodiments include addedmaterials to reduce intercellular effects. We have found that additionof about 5mM EDTA to the wash liquid or resuspension buffer (PBS withfetal calf serum or BSA of density about 1.0015 g/mL or about 0.1% toabout 2% concentration) prevents formation of an indeterminate cellpopulation (possibly degranulated granulocytes) that appear adjacentpopulations of monocytes and granulocytes in white blood cell samples.

A protocol may use different wash liquids during different portions ofthe wash process. A protocol may also use a resuspension buffer that isdifferent from wash liquids in the final resuspension step. Thecontroller may select the desired added liquids according to a protocolby switching appropriate ones of valves 74 to connect a reservoir 70containing the desired liquid to bidirectional pump 68 in a mannerfamiliar to those skilled in the art of fluid handling.

All steps described are controlled by the controller. The controllerreceives its instructions from a software program stored in its programmemory. The controller (or a second computer operating as a userinterface) accepts user instruction and (transmits the instructions tothe controller, which) sequences the various mechanical componentsaccording to the selected protocol.

An exemplary cell washing process is illustrated diagrammatically inFIGS. 3A-3F and in the flow chart of FIG. 4 . In general, a methodaccording to an embodiment of the invention may include a method ofwashing cells from a sample including cells suspended in a liquid, themethod comprising: dispensing the sample into a vessel including a bodydefining a cavity and a pocket, the pocket extending radially outwardrelative to an inner wall surface defining the cavity; rotating thevessel about the axis at a first speed; displacing the cells into thepocket; sedimenting the cells within the pocket; and withdrawing atleast part of the liquid.

In step 102 and as illustrated in FIG. 3A, probe 54 dispenses a sample140 into vessel 10. The vessel 10 may include the same or differentfeatures as described above. Then, probe 54 aspirates an aliquot ofsample 140, descends into vessel 10, and deposits the aliquot of sample140 at or near the bottom of the vessel 10. Elevator 56 then withdrawsprobe 54. FIG. 3A shows the position of sample 140 in vessel 10 afterthe transfer is completed.

In step 104 and as illustrated in FIG. 3B, the controller rotates (e.g.,spins) vessel 10 at a first speed sufficient to produce at least about100 or 250 g at the inner wall of pocket 34. Once spinning, conduit 18dispenses a wash liquid 144 at a high rate (about 1 mL per second). Thevolume is sufficient to displace the some or the entirety of the initialsample 140 into pocket 34. In some embodiments, the high injection ratealong wall 28 provides inertia to displace sample 140 rather thancompletely mix with it. In this and other steps involving liquidaddition by conduit 18, controller 20 sequences operations ofbidirectional pump 68 and any appropriate valve 74 to direct liquid fromreservoir 70 through tube 64 and out tip 66.

In step 106 and as illustrated in FIG. 3C, spin continues at the firstspeed for about five seconds to sediment cells 142 to the inner wallsurface portion 37A of pocket 34. Then controller 20 reduces spin speedto a lower wash speed.

In step 108 and as illustrated in FIG. 3D, conduit 18 injects additionalwash fluid 144 at a lower flow rate (about 200 µL per second) to fillvessel 10 (bring the edge of the fluid-free margin to about the radiusof circular opening 44 at top end 38. Shear force pulses, generated byinteraction of tube 64 with the liquid and the fluid air boundary duringfluid injection, helps scrub cells 142 confined against the wall ofpocket 34. The added wash fluid 144 moves through the cells vertically,so that the direction of wash fluid flow is substantially perpendicularto the direction of centrifugal force holding cells 142 in pocket 34. Abenefit of this perpendicular flow is that it reduces unswept volumesamong the cells 142. The shear force pulses may be modulated by alteringthe rotation rate and fluid injection speed. In some embodimentscontroller 20 varies the rotation rate to move the fluid-free margin upor down through the pocket to scrub the cells retained therein.

In step 114 and as illustrated in FIG. 3E, conduit 18 aspirates liquidat a high rate, ramping down the rotation rate while aspirating tothicken the layer of liquid near tip 66. In this and other stepsinvolving liquid removal by conduit 18, controller 20 sequencesoperations of bidirectional pump 68 and any appropriate valve 74 todirect liquid into tip 66, through tube 64, and to waste 72. Minimumspin rate produces a force of about 25 x g on cells 142 to retain cells142 in pocket 34. Conduit 18 then adds wash liquid so that the totalvolume approximates the initial sample volume. Controller 20 transientlystops or reverses rotation to resuspend the cells from pocket 34.Reversal may include two changes of direction (the second restores theoriginal direction of rotation). These changes in rotation take place ina relatively short time-less than about one second. Such rapid changesproduce high tangential accelerations that act as inertial forces on thecontents of vessel 10. The inertial forces increase with radius so thatthe cells sedimented in pocket 34 experience the highest inertialforces. These inertial forces, in conjunction with viscous and buoyanteffects in the liquid, serve to agitate the cells and intermix them withadjacent portions of the liquid.

In step 112, controller 20 repeats steps 106 through 110 a number oftimes. More cycles result in greater wash efficacy. In some embodiments,controller 20 sequences two or three repetitions of steps 106 through110. During the final wash the amount of liquid added is adjusted tosuspend the particles at the desired concentration for cytometry orother subsequent processing. The volume of added liquid may be less thanthe original volume of the sample.

In step 110 and as illustrated in FIG. 3F, rotation stops, and probe 54descends to near the bottom of vessel 10 and aspirates the washed samplefor analysis or further processing elsewhere. The system then cleansvessel 10 to prepare for the next use.

An exemplary vessel cleaning process 120 is illustrated diagrammaticallyin FIGS. 5A-5F and in the flow chart of FIG. 6 .

In step 122 and as illustrated in FIG. 5A, conduit 18 adds rinse liquidrapidly without spinning vessel 10.

At step 124, vessel 10 spins, then conduit 18 aspirates rinse liquidduring the spin. Controller 20 varies the spin speed to move theparabolic fluid-free margin up and down through vessel 10 as illustratedin FIGS. 5B-5D.

At step 126 and as illustrated in FIG. 5E, conduit 18 aspirates allremaining accessible fluid as rotation slows.

At step 128, controller 20 adds rinse liquid thorough conduit 18 andrepeats steps 124 through 126 one or more times. In some embodiments,the controller sequences two or three repetitions of steps 124 through126. More cycles result in improved vessel cleanliness and reducedcarryover.

At step 130 and as illustrated in FIG. 5 f ), probe 54 descends tobottom of vessel 10 and aspirates all remaining accessible liquid.Vessel 10 is then ready for reuse.

Example 1: Experimental Wash

Each of Examples 3 - 10 use the vessel and protocol embodiments below:

Vessel 10 is about 2 inches tall and has upper and lower portions eachabout 0.4 inches tall and a pocket about 1 inch tall (the axial extentor height). Upper and lower portions have cylindrical sections withinside diameter of about 0.3125 inches; the pocket has inside diameterof about 0.4125 inches. Thus, in this embodiment, the pocket forms acylindrical shell with depth of about 0.05 inches and maximum radius ofabout 0.206 inches. The shell depth is about one eighth of the shellouter diameter. The shell depth is about one twentieth of the axialextent of the pocket. The diameter of the central hole is about 0.157inches. The inside walls of the upper and lower portions taper away fromthe pocket by terminating in hemispherical sections.

The example protocol includes the following steps, as modified wherenoted by experimental variations:

-   a) using probe, add 100 µL whole blood mixed with 400 µL red cell    lysing agent and labels as required by each experiment;-   b) spin at 6460 rpm (to produce about 100 or 250 x g at pocket    wall);-   c) using conduit, add 500 µL wash liquid at 1 mL/sec;-   d) sediment cells to pocket wall for 5 sec;-   e) using conduit, add 2 mL wash liquid at 200 µL/sec;-   f) using conduit, remove 2.4 mL liquid at 1 mL/sec while decreasing    spin speed linearly to 2000 rpm (to produce about 25 x g at pocket    wall);-   g) using conduit, add 400 µL wash liquid and reverse rotor twice for    0.5 seconds to resuspend cells;-   h) repeat steps e), f), and g) for number of times as required by    each experiment. After last repeat of step f), replace wash liquid    in step g) with resuspension buffer and stop rotation; and-   i) using probe, aspirate washed cells and analyze in a flow    cytometer.

Example 2: traditional wash

-   a) manually load 100 µL whole blood mixed with 400 µL red cell    lysing agent and labels as required by each experiment into 12 x 75    mm polypropylene tubes;-   b) add 2 mL wash liquid; mix by inversion;-   c) spin 500 x g for 5 min to pellet cells;-   d) aspirate supernatant by pipette;-   e) repeat steps b) through d) twice; and-   f) resuspend pellet in 400 µL resuspension buffer and mix by    inversion.

Example 3: Biological Equivalency-Scatter

FIG. 7 compares scatter plots of white blood cell recovery usingembodiments of the device and method of the invention compared totraditional wash and to no wash. Upper row plots forward scatter (FS)against side scatter (SS) for unwashed cells, traditionally washedcells, and cells washed according to Example 1 (test method). Lower rowplots CD45 (labeled with Krome Orange) against SS. Krome Orange is atrademark of Beckman Coulter, Inc. Light scatter and representation ofWBC populations using test method are comparable to traditionally washedand to unwashed cells.

Example 4: Biological Equivalency - Five-part differential

FIG. 8 compares scatter plots of white blood cell recovery using thetest method compared to traditional wash and to no wash. Upper row plotsCD45 (labeled with FITC) against SS for unwashed cells, traditionallywashed cells, and cells washed according to test method. Lower row plotsCD14 (labeled with PE-Cy7) against SS. Calculated values below each setof plots shows recoveries for neutrophils, monocytes, lymphocytes,eosinophils, and basophils is comparable to traditional cell wash and tono wash.

Example 5: Biological Equivalency-Platelets

FIG. 9 compares scatter plots of CD14 (labeled with PE-Cy7) against CD41(a platelet marker here labeled with APC) using the test method comparedto traditional wash and to no wash. Plots were gated on the rectangularregion marked Monos in the lower plots of FIG. 8 . Note the considerablereduction in density of monocytes showing CD41 binding, especially ascompared to the traditional cell wash method. Test method included 5 mMEDTA in wash buffer. Platelets attachment to monocytes is significantlydecreased for the test method.

Example 6: Biological Equivalency- Viability

FIG. 10 compares scatter plots of analysis of white blood cell viabilityusing the test method compared to traditional wash and to no wash. Theplots display signal from 7-Aminoactinomycin D (7AAD) against SS. 7AADbinds to dead cells but not live cells. Calculated cell viability showsinsignificant decreases compared to traditional cell wash and isacceptable for flow cytometry analysis.

Example 7: Biological Equivalency: kappa / lambda separation

FIG. 11 compares scatter plots of B cells displaying lambda (labeledwith PE) against kappa (labeled with FITC). The diagonal lines separatekappa positive B cells from lambda positive B cells using the testmethod compared to traditional wash. The calculated values are signalseparation expressed as signal to noise ratios. These are calculated asKappa = KX-median / LX-median and Lambda = LY-median / KY-median. Gatedvalues for Kappa and Lambda are comparable to traditional wash.

Example 8: Biological Equivalency: Total Cell Recovery

FIG. 12 compares scatter plots of ungated white blood cells for analysisof total cell recovery using the test method compared to traditionalwash and to no wash. The plots display signal from CD45 (labeled withFITC) against SS as in Example 4. The calculated values are cellsrecovered compared to the no wash case.

Example 9: Biological Equivalency: Cell Subset Recovery I

FIG. 13 compares histograms of T-cell subset recovery using the testmethod compared to traditional wash and to no wash. The histogramsdisplay bins of CD3 signal (labeled with PE-Cy5). Subset cell recoveryfor the test method (gated from WBCs labeled with Beckman Coulter, Inc.Tetra Panel) is comparable to traditional and no wash.

Example 9: Biological Equivalency: Cell Subset Recovery II

FIG. 14 compares scatter plots of B cells and NK cell recovery using thetest method compared to traditional wash and to no wash. The plotsdisplay signal from CD56 (labeled with Rhodamine) against CD19 (labeledwith ECD) (gated from WBCs labeled with Beckman Coulter, Inc. TetraPanel). B-cell and NK cell recovery for the test method is comparable totraditional and no wash.

FIGS. 15 and 16 show an embodiment that includes two probes, a dispenseprobe and an aspiration probe. There are several benefits to having twoconduits such as two probes. If a single probe is used, then one probehas to dispense clean buffer and remove waste. Therefore, one benefit ofusing a system with two probes is to minimize contamination betweenadjacent runs. In other words, clean buffer can be contaminated from theprevious run’s waste. A second benefit, is that the two probes can beplaced at different locations. The dispense probe (which can dispenseclean buffer) can be located at the top of a vessel while the removalprobe (which can dispense waste) is located at the bottom of the vessel.At the end of the run, a small amount of buffer is dispensed out of theupper probe to clean the inner walls as the buffer falls to the bottomof the vessel. This helps with recovery of the blood cells during ablood purification process.

FIG. 15 shows a partial side cross-sectional view of an assemblyaccording to an embodiment of the invention. FIG. 15 shows a vessel 200,which can have the configuration of the previously described vesselembodiments. A dispense probe 202 and an aspiration probe 204 and theirdistal ends are disposed within the vessel 200. The aspiration probe 204is linear and extends towards a bottom of the vessel 200, and can beconfigured to remove waste buffer. The dispense probe 202 has at its enda 90 degree bend to add wash buffer to the pocket 206 and any materialthat might be in the pocket 206. The aspiration probe 204 may also havea 90 degree bend at its distal end, or it could be linear. The bend ismore clearly illustrated in FIG. 16 . An end of the aspiration probe 204can be just below the pocket 206. Although the ends of the dispenseprobe 202 and the aspiration probe 204 are shown to be orthogonal toeach other in FIGS. 15 and 16 , in other embodiments, they may be withinthe same plane, or possibly aligned in a similar manner.

FIG. 16 shows a top cross-sectional view of the assembly shown in FIG.15 . FIG. 16 shows a top down view of the distal end portions of thedispense probe 202 and the aspiration probe 204. As shown in FIG. 16 ,the long axes of the dispense probe 202 and the aspiration probe 204 areoff center with respect to a center axis of the vessel.

Some exemplary wash routines that can be performed using embodiments ofthe invention can be described as follows. One wash routine is a “samplewash,” where a specimen such as blood (e.g., 100 uL of blood) is washed.The second wash routine can be a “lyse wash,” where the non-cellularproducts of cell lysis are removed from the blood sample. The thirdroutine is a “bulk wash,” where larger amounts (e.g., 500 uL) of bloodare washed. In some embodiments, a full workflow may include the firstwash routine (sample wash) followed by the second wash routine (lysewash).

Sample Wash (e.g., of a Single 100 uL Specimen)

Step 1. Dispense a specimen (e.g. blood) into a vessel.

Step 2. Dispense a fresh wash buffer into the vessel with a dispenseprobe located at an upper region of the vessel.

Step 3. Rotate the vessel back-and-forth to cause the specimen tovortex.

Step 4. Spin the vessel at a maximum speed to pellet components of thespecimen into the vessel pocket.

Step 5. Remove the waste buffer using the aspiration probe located nearthe bottom of the vessel as the speed of rotation of the vessel isreduced.

Step 6. Perform steps 2 to 4 a total of four or more times.

Step 7. Spin the vessel back-and-forth in intervals. Each interval is aslower speed than the previous one. This is to dislodge any cells thatmight be adhered to the walls.

Step 8. Dispense a small amount of buffer into the vessel using theupper probe to clean the vessel walls while the tube spins slowly.

Lyse Wash

Step 1. Transfer the lysed solution with an external pipette into thevessel. The lyse solution may include the specimen and a lysing agent.For example, the lyse solution may include 100 uL of blood and 2.0 mL ofIOTest Lyse (an ammonium chloride-based erythrocyte lysing solutionavailable from Beckman Coulter, Inc.). The total volume may be more than2.0 ml. (e.g., 2.1 mL).

Step 2. Immediately spin the vessel at maximum speed to pellet the bloodinto the pocket.

Step 3. Remove the lyse supernatant (with the lower aspiration probe) asthe rotational speed of the vessel is reduced.

Step 4. Stop the rotation of the vessel.

Step 5. Dispense fresh wash buffer with the upper dispense probe.

Step 6. Spin the vessel back and forth to create a vortex in thespecimen.

Step 7. Spin the vessel at maximum speed to pellet components of thespecimen into the vessel pocket.

Step 8. Remove the waste buffer using the lower aspiration probe as therotational speed of the vessel is reduced.

Step 9. Spin the vessel back and forth in intervals. Each interval is ata slower speed than the previous one. This can be used to dislodge anycells that might be adhered to the walls.

Step 10. Dispense a small amount of buffer using the upper dispenseprobe to clean the vessel walls while the vessel is spins slowly.

Bulk Wash (500 uL of Specimen)

1. Use the same procedure as Sample Wash except the durations of eachstep are longer

Although aspects of the present specification are highlighted byreferring to specific embodiments, these disclosed embodiments are onlyillustrative of the principles of the subject matter. The inventionencompasses any combination of features in described embodiments unlessotherwise indicated or clearly contradicted by context.

Unless otherwise indicated, all numbers expressing a characteristic,item, quantity, parameter, property, term, and so forth used in thepresent specification and claims are to be understood as being modifiedin all instances by the term “about.” As used herein, the term “about”means that the characteristic, item, quantity, parameter, property, orterm so qualified encompasses a range of plus or minus twenty percentabove and below the value of the stated characteristic, item, quantity,parameter, property, or term. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the specification andattached claims are approximations that may vary.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the present invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. All methods described herein can be performed in any suitableorder unless otherwise indicated or clearly contradicted by context. Theuse of examples, or exemplary language (e.g., “such as”) is intendedmerely to better illuminate the present invention and not as alimitation on the scope of the invention. No language in the presentspecification should be construed as indicating any non-claimed elementessential to the practice of the invention.

1-15. (canceled)
 16. A method of washing cells from a sample includingcells suspended in a liquid, the method comprising: a) dispensing thesample into a vessel including a body defining a cavity and a pocket,the pocket extending radially outward relative to an inner wall surfacedefining the cavity; b) rotating the vessel about an axis of the vesselat a first speed; c) displacing the cells into the pocket; d)sedimenting the cells within the pocket; and e) withdrawing at leastpart of the liquid.
 17. The method of claim 16, wherein washed cells areformed after step e) and wherein the step of displacing includes: addinga first aliquot of a wash liquid through a conduit extending into thevessel and terminating below the pocket, and wherein the step ofwithdrawing includes aspirating through the conduit.
 18. The method ofclaim 17, further comprising: f) adding a second aliquot of the washliquid through the conduit.
 19. The method of claim 17, furthercomprising resuspending the washed cells.
 20. The method of claim 19,wherein the step of resuspending includes adding a resuspension liquidto the vessel and stopping rotating.
 21. The method of claim 19, whereinthe step of resuspending includes reversing a direction of rotation ofthe vessel.
 22. The method of claim 19, wherein the cavity has a volumebelow the pocket, and the sample has a sample volume less than thevolume below the pocket.
 23. The method of claim 22, wherein the samplevolume is less than about 0.7 times the volume below the pocket.
 24. Themethod of claim 20, wherein the resuspension liquid has a resuspensionvolume less than the sample volume.
 25. The method of claim 16, furthercomprising rotating the vessel at a second speed during the withdrawingstep.
 26. The method of claim 20, wherein rotating the vessel at asecond speed produces a force in the pocket of at least 25 x g.
 27. Themethod of claim 16, wherein rotating the vessel at the first speedproduces a force in the pocket of at least about 100 x g.
 28. The methodof claim 18, further comprising repeating steps e) and f).
 29. Themethod of claim 18, further comprising g) cleaning the vessel.
 30. Themethod of claim 29, wherein step g) includes: i. adding a rinse liquid;ii. rotating the vessel at a third speed; and iii. aspirating the rinseliquid through the conduit.
 31. The method of claim 29, wherein the stepof cleaning the vessel includes varying a speed of rotation during thestep of aspirating the rinse liquid.
 32. The method of claim 20, whereinthe wash liquid or the resuspension liquid includes about 5 mM EDTA. 33.The method of claim 16, further comprising: adding a rinse liquid intothe vessel using a dispense probe terminating within the vessel. 34.Method of claim 33, further comprising, aspirating at least part of therinse liquid using an aspiration probe terminating within the vessel.35. The method of claim 34, wherein the aspiration probe terminates at alower point within the vessel than the dispense probe. 36-39. (canceled)